Methods for fabricating multi-component devices for molecular biological analysis and diagnostics

ABSTRACT

A method for manufacturing a multicomponent flip-chip device is disclosed. The device includes a chip disposed adjacent to a substrate, the substrate including a via therethrough. The device is adapted to receive a fluid to be placed on the substrate, wherein the fluid then flows through the via down to the chip. The chip includes a sealant free region and a sealant receiving region. The method includes the steps of placing a chip adjacent to a substrate. Light is exposed to the substrate and through the via, down onto the surface of the chip. A light-curable, wickable sealant is applied to the interface between the substrate and the chip, wherein the light at least partially cures the sealant to preclude the sealant from flowing to the sealant free region. Additional curing of sealant may also be performed.

RELATED APPLICATION INFORMATION

This application is a continuation-in-part application of applicationSer. No. 09/026,618, filed Feb. 20, 1998, now U.S. Pat. No. 6,099,803entitled “Advance Active Electronic Devices for Molecular BiologicalAnalysis and Diagnostics and Methods for Manufacture of Same”, now U.S.Pat. No. 6,099,803, which is a continuation-in-part of application Ser.No. 08/753,962, filed Dec. 4, 1996, entitled “Laminated Assembly forActive Bioelectronic Devices”, which is a continuation-in-part of Ser.No. 08/534,454, filed Sep. 27, 1995, entitled “Apparatus and Methods forActive Programmable Matrix Devices”, now issued as U.S. Pat. No.5,849,486, which is a continuation-in-part of application Ser. No.08/304,657, filed Sep. 9, 1994, entitled, as amended, “MolecularBiological Diagnostic Systems Including Electrodes”, now issued as U.S.Pat. No. 5,632,957, continued as Ser. No. 08/859,644, which is acontinuation-in-part of application Ser. No. 08/271,882, filed Jul.7,1994 filled May 20, 1997, entitled “Control System for ActiveProgrammable Electronic Microbiology System” entitled, as amended,“Methods for Electronic Stringency Control for Molecular BiologicalAnalysis and Diagnostics”, now U.S. Pat. No. 6,017,696, which is acontinuation-in-part of application Ser. No. 08/146,504, filed Nov. 1,1993, entitled, as amended, “Active Programmable Electronic Devices forMolecular Biological Analysis and Diagnostics”, now issued as U.S. Pat.No. 5,605,662, continued as application Ser. No. 08/725,976 filed Oct.4, 1996, entitled “Methods for Electronic Synthesis of Polymers”, nowU.S. Pat. No. 5,929,208 and application Ser. No. 08/709,358, filed Sep.6, 1996, entitled “Apparatus and Methods for Active Biological SamplePreparation”now U.S. Pat. No. 6,129,828; and is related to applicationSer. No. 08/677,305, filled Jul. 9, 1996, entitled “Multiplexed ActiveBiological Array”, now U.S. Pat. No. 5,965,452; and is also related toapplication Ser. No. 08/846,876, filed May 1, 1997, entitled “ScanningOptical Detection System”, all incorporated herein by reference as iffully set forth herein.

This application is also related to the following applications filed oneven date herewith, entitled “Advanced Active Electronic DevicesIncluding Collection Electrodes for Molecular Biological Analysis andDiagnostics”, “Multicomponent Devices for Molecular Biological Analysisand Diagnostics”, “Devices for Molecular Biological Analysis andDiagnostics Including Waveguides”, and “Advanced Active Circuits andDevices for Molecular Biological Analysis and Diagnostics”, all of whichare incorporated herein by reference.

1. Field of the Invention

These invention relates to methods of manufacture and devices useful inperforming active biological operations. More particularly, theinventions relate to devices and methods for manufacture of such devicescontaining active electrodes especially adapted for electrophoretictransport of nucleic acids, their hybridization and analysis.

2. Background of the Invention

Molecular biology comprises a wide variety of techniques for theanalysis of nucleic acid and protein. Many of these techniques andprocedures form the basis of clinical diagnostic assays and tests. Thesetechniques include nucleic acid hybridization analysis, restrictionenzyme analysis, genetic sequence analysis, and the separation andpurification of nucleic acids and proteins (See, e. g., J. Sambrook, E.F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, 1989).

Most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugations, electrophoresis) on a large number ofsamples. They are often complex and time consuming, and generallyrequire a high degree of accuracy. Many a technique is limited in itsapplication by a lack of sensitivity, specificity, or reproducibility.For example, these problems have limited many diagnostic applications ofnucleic acid hybridization analysis.

The complete process for carrying out a DNA hybridization analysis for agenetic or infectious disease is very involved. Broadly speaking, thecomplete process may be divided into a number of steps and substeps. Inthe case of genetic disease diagnosis, the first step involves obtainingthe sample (blood or tissue). Depending on the type of sample, variouspre-treatments would be carried out. The second step involves disruptingor lysing the cells, which then release the crude DNA material alongwith other cellular constituents. Generally, several sub-steps arenecessary to remove cell debris and to purify further the crude DNA. Atthis point several options exist for further processing and analysis.One option involves denaturing the purified sample DNA and carrying outa direct hybridization analysis in one of many formats (dot blot,microbead, microplate, etc. ). A second option, called Southern blothybridization, involves cleaving the DNA with restriction enzymes,separating the DNA fragments on an electrophoretic gel, blotting to amembrane filter, and then hybridizing the blot with specific DNA probesequences. This procedure effectively reduces the complexity of thegenomic DNA sample, and thereby helps to improve the hybridizationspecificity and sensitivity. Unfortunately, this procedure is long andarduous. A third option is to carry out the polymerase chain reaction(PCR) or other amplification procedure. The PCR procedure amplifies(increases) the number of target DNA sequences relative to non-targetsequences. Amplification of target DNA helps to overcome problemsrelated to complexity and sensitivity in genomic DNA analysis. All theseprocedures are time consuming, relatively complicated, and addsignificantly to the cost of a diagnostic test. After these samplepreparation and DNA processing steps, the actual hybridization reactionis performed. Finally, detection and data analysis convert thehybridization event into an analytical result.

The steps of sample preparation and processing have typically beenperformed separate and apart from the other main steps of hybridizationand detection and analysis. Indeed, the various substeps comprisingsample preparation and DNA processing have often been performed as adiscrete operation separate and apart from the other substeps.Considering these substeps in more detail, samples have been obtainedthrough any number of means, such as obtaining of full blood, tissue, orother biological fluid sample. In the case of blood, the sample isprocessed to remove red blood cells and retain the desired nucleated(white) cells. This process is usually carried out by density gradientcentrifugation. Cell disruption or lysis is then carried out on thenucleated cells to release DNA, preferably by the technique ofsonication, freeze/thawing, or by addition of lysing reagents. Crude DNAis then separated from the cellular debris by a centrifigation step.Prior to hybridization, double-stranded DNA is denatured into singlestranded form. Denaturation of the double-stranded DNA has generallybeen performed by the techniques involving heating (>Tm), changing saltconcentration, addition of base (NaOH), or denaturing reagents (urea,formamide, etc.).

Nucleic acid hybridization analysis generally involves the detection ofa very small number of specific target nucleic acids (DNA or RNA) withan excess of probe DNA, among a relatively large amount of complexnon-target nucleic acids. The substeps of DNA complexity reduction insample preparation have been utilized to help detect low copy numbers(i.e. 10,000 to 100,000) of nucleic acid targets. DNA complexity isovercome to some degree by amplification of target nucleic acidsequences using polymerase chain reaction (PCR). (See, M. A. Innis etal, PCR Protocols: A Guide to Methods and Applications, Academic Press,1990). While amplification results in an enormous number of targetnucleic acid sequences that improves the subsequent direct probehybridization step, amplification involves lengthy and cumbersomeprocedures that typically must be performed on a stand alone basisrelative to the other substeps. Substantially complicated and relativelylarge equipment is required to perform the amplification step.

The actual hybridization reaction represents one of the most importantand central steps in the whole process. The hybridization step involvesplacing the prepared DNA sample in contact with a specific reporterprobe, at a set of optimal conditions for hybridization to occur to thetarget DNA sequence. Hybridization may be performed in any one of anumber of formats. For example, multiple sample nucleic acidhybridization analysis has been conducted on a variety of filter andsolid support formats (See G. A. Beltz et al., in Methods in Enzvmology,Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press,New York, Chapter 19, pp. 266-308, 1985). One format, the so-called “dotblot” hybridization, involves the non-covalent attachment of target DNAsto filter, which are subsequently hybridized with a radioisotope labeledprobe(s). “Dot blot” hybridization gained wide-spread use, and manyversions were developed (see M. L. M. Anderson and B. D. Young, inNucleic Acid Hvbridization—A Practical Approach, B. D. Hames and S. J.Higgins, Eds., IRL Press, Washigton, D. C. Chapter 4, pp. 73-111, 1985).It has been developed for multiple analysis of genomic mutations (D.Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for thedetection of overlapping clones and the construction of genomic maps (G.A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).

New techniques are being developed for carrying out multiple samplenucleic acid hybridization analysis on micro-formatted multiplex ormatrix devices (e.g., DNA chips) (see M. Barinaga, 253 Science, pp.1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). Thesemethods usually attach specific DNA sequences to very small specificareas of a solid support, such as micro-wells of a DNA chip. Thesehybridization formats are micro-scale versions of the conventional “dotblot” and “sandwich” hybridization systems.

The micro-formatted hybridization can be used to carry out “sequencingby hybridization”(SBH) (see M. Barinaga, 253 Science, pp. 1489, 1991; W.Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of allpossible n-nucleotide oligomers (n-mers) to identify n-mers in anunknown DNA sample, which are subsequently aligned by algorithm analysisto produce the DNA sequence (R. Drmanac and R. Crkvenjakov, YugoslavPatent Application #570/87,1987; R. Drmanac et al., 4 Genomics, 114,1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1992; andR. Drmanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13,1993).

There are two formats for carrying out SBH. The first format involvescreating an array of all possible n-mers on a support, which is thenhybridized with the target sequence. The second format involvesattaching the target sequence to a support, which is sequentially probedwith all possible n-mers. Both formats have the fundamental problems ofdirect probe hybridizations and additional difficulties related tomultiplex hybridizations.

Southern, United Kingdom Patent Application GB 8810400, 1988; E. M.Southern et al., 13 Genomics 1008, 1992, proposed using the first formatto analyze or sequence DNA. Southern identified a known single pointmutation using PCR amplified genomic DNA. Southern also described amethod for synthesizing an array of oligonucleotides on a solid supportfor SBH. However, Southern did not address how to achieve optimalstringency condition for each oligonucleotide on an array.

Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used thesecond format to sequence several short (116 bp) DNA sequences. TargetDNAs were attached to membrane supports (“dot blot” format). Each filterwas sequentially hybridized with 272 labeled 10-mer and 11-meroligonucleotides. A wide range of stringency condition was used toachieve specific hybridization for each n-mer probe; washing timesvaried from 5 minutes to overnight, and temperatures from 0° C. to 16°C. Most probes required 3 hours of washing at 16° C. The filters had tobe exposed for 2 to 18 hours in order to detect hybridization signals.The overall false positive hybridization rate was 5% in spite of thesimple target sequences, the reduced set of oligomer probes, and the useof the most stringent conditions available.

A variety of methods exist for detection and analysis of thehybridization events. Depending on the reporter group (fluorophore,enzyme, radioisotope, etc. ) used to label the DNA probe, detection andanalysis are carried out fluorimetrically, colorimetrically, or byautoradiography. By observing and measuring emitted radiation, such asfluorescent radiation or particle emission, information may be obtainedabout the hybridization events. Even when detection methods have veryhigh intrinsic sensitivity, detection of hybridization events isdifficult because of the background presence of non-specifically boundmaterials. A number of other factors also reduce the sensitivity andselectivity of DNA hybridization assays.

Attempts have been made to combine certain processing steps or substepstogether. For example, various microrobotic systems have been proposedfor preparing arrays of DNA probe on a support material. For example,Beattie et al., in The 1992 San Diego Conference: Genetic Recognition,November, 1992, used a microrobotic system to deposit micro-dropletscontaining specific DNA sequences into individual microfabricated samplewells on a glass substrate.

Generally, the prior art processes have been extremely labor and timeintensive. For example, the PCR amplification process is time consumingand adds cost to the diagnostic assay. Multiple steps requiring humanintervention either during the process or between processes issuboptimal in that there is a possibility of contamination and operatorerror. Further, the use of multiple machines or complicated roboticsystems for performing the individual processes is often prohibitiveexcept for the largest laboratories, both in terms of the expense andphysical space requirements.

Attempts have been made to enhance the overall sample introduction, tosample preparation analysis process. Given the relatively small volumeof sample material which is often times available, improved processesare desired for the efficient provisions of sample, transport of sampleand effective analysis of sample. While various proposals have beenadvanced, certain systems enjoy relative advantages in certaincircumstances.

Yet another area of interest is in the electrical addressing ofrelatively large arrays. As array grow relatively large, the efficientoperation of the system becomes more of a consideration. Efficientinterfacing of an array based system with electrical connectionsoff-chip raise pin or contact limitation issues. Further, constraintsregarding effective chip or array size present issues regarding theselection of components, and the size of them, for inclusion on the chipor substrate. Often times, various selections must be made to provide aneffective optimization of advantages in the overall design.

One proposed solution for the control of an array of electrodesutilizing less than one individual dedicated connection per electrode ortest site is provided in Kovacs U.S. patent application Ser. No.08/677,305, entitled “Multiplexed Active Biological Array”, filed Jul.9, 1996, incorporated herein as if fully set forth herein. The array isformed of a plurality of electrode sites, a typical electrode siteincluding an electrode, a driving element coupled to the electrode forapplying an electrical stimulus to the electrode and a local memorycoupled to the driving element for receiving and storing a signalindicative of a magnitude of the electrical stimulus to be applied tothe electrode. Multiple embodiments are disclosed for selectivelycoupling a value signal through coaction of a row line and a column linefor storage in the local memory. In this way, the values at the variouselectrodes in the array may differ from one another.

In Fiaccabrino, G. C., et al., “Array of Individual AddressableMicroelectrodes”, Sensors and Actuators B, 18-19, (1994) 675-677, anarray of n² electrodes are connected to two n pins, plus 2 additionalpins for signal output and bulk bias. The row and column signals driveseries connected transistors to provide a single value to a workingelectrode. This system does not enable the switching of two or moreelectrodes simultaneously at different potentials.

In Kakerow, R. et al., “A Monolithic Sensor Array of IndividuallyAddressable Microelectrodes”, Sensors and Actuators A, 43 (1994)296-301, a monolithic single chip sensor array for measuring chemicaland biochemical parameters is described. A 20×20 array of individuallyaddressable sensor cells is provided. The sensor cells are seriallyaddressed by the sensor control unit. One horizontal and one verticalshift register control selection of the sensor cells. Only one sensorcell is selected at a time. As a result, multiple sites may not beactivated simultaneously.

Yet another concern is the ability to test an electronic device prior toapplication of a conductive solution on the device. As devices or chipsbecome more complicated, the possibility of a manufacturing or processerror generally increases. While visual inspection of circuitry may beperformed, further testing may ensure an operational device is providedto the end user.

As is apparent from the preceding discussion, numerous attempts havebeen made to provide effective techniques to conduct multi-step,multiplex molecular biological reactions. However, for the reasonsstated above, these techniques are “piece-meal”, limited and have noteffectively optimized solutions. These various approaches are not easilycombined to form a system which can carry out a complete DNA diagnosticassay. Despite the long-recognized need for such a system, nosatisfactory solution has been proposed previously.

SUMMARY OF THE INVENTION

Methods of manufacture and apparatus adapted for advantageous use inactive electronic devices utilized for biological diagnostics aredisclosed. Specifically, various layouts or embodiments, including theselection of components, are utilized in advantageous combination toprovide useful devices. Various structures, shapes and combinations ofelectrodes coact with various applied signals (voltages, currents) so asto effect useful preparation, transport, diagnosis, and analysis ofbiological or other electrically charged material. Various advantageousprotocols are described.

In a first preferred embodiment, an electronic device for performingactive biological operations comprises in combination a supportsubstrate, an array of micro-locations disposed on the substrate, afirst collection electrode disposed on the substrate, first and secondfocusing electrodes disposed on the substrate, the first and secondelectrodes being disposed at least in part adjacent the array ofmicrolocations, the distance between the first and second electrodesadjacent the array preferably being smaller than the distance betweenthe first and second electrodes in yet another region disposed away fromthe array, and counter electrodes disposed on the substrate. In oneimplementation, a “V” or “Y” configuration is utilized, which serves tofocus charged biological material into a desired region. Preferably, thefocusing electrodes have a proximal end disposed near or adjacent thearray of microlocations, and a remote portion disposed away from thearray. The distance between the proximal ends of the first and secondelectrode is less than the distance between the proximal ends of thefirst and second electrode.

In operation of this embodiment, a solution containing DNA or otherbiological material to be interrogated is provided to the device, abovethe substrate. As a typical initial step, the concentration electrodeand return electrodes are activated so as to transport and concentratethe charged biological materials onto or near the concentration region.In the preferred embodiment, the concentration electrode and the returnelectrode or electrodes interrogate a relatively large volume of thesample. Typically, the collection electrode and counter electrodes aredisposed on the substrate so that the electrophoretic lines of force aresignificant over substantially all of the flow cell volume. By way ofexample, the concentration and return electrodes may be disposed nearthe periphery of the footprint of the flow cell. In yet anotherembodiment, they are maybe disposed at substantially opposite ends ofthe flowcell. In yet another embodiment, the return electrodesubstantially circumscribes the footprint of the flow, with a centrallydisposed collection electrode. Effective interrogation of the samplewithin the flow cell is one desired result. Once the sample has beenconcentrated, the focusing electrodes may be operated so as to funnel orfurther focus the materials towards the array of microlocations. Asmaterials move from the concentration electrode towards the array, thedecreasing spacing between the first and second focusing electrodesserves to concentrate the analytes and other charged material into asmaller volume. In this way, a more effective transportation ofmaterials from a relatively larger concentration electrode region to arelatively smaller microelectrode array region may be achieved.

It yet another optional aspect of this embodiment of this invention, oneor more transport electrodes are provided, the transport electrodesbeing disposed on the substrate, and positioned between the firstcollection electrode and the array. In the preferred embodiment, thereare at least two transport electrodes, and further, the transportelectrodes are of a different size, preferably wherein the ratio oflarger to smaller is at least 2:1. In this way, the relatively largearea subtended by the collection electrode may be progressively moved tosmaller and smaller locations near the analytical region of the device.This arrangement both aids in transitioning from the relatively largearea of the collection electrode, but the stepped nature of theembodiment reduces current density mismatches. By utilizing a stepped,preferably monotonically stepped size reduction, more effectivetransportation and reduced burnout are achieved.

In yet another embodiment of device, an electronic device for performingbiological operations comprises a support substrate, an array ofmicrolocations disposed on the substrate, the array being formed withina region, the region including a first side and an opposite side, afirst collection electrode disposed on the substrate adjacent the array,and a second collection electrode disposed on the substrate, adjacentthe array, the first and second collection electrodes being at least inpart on the opposite side of the region. In the preferred embodiment,the collection electrodes have an area at least 80% of the area of theregion of the array. In this way, the sample may be collected in arelatively large area adjacent the region containing microlocations,from which the DNA or other charged biological materials may be providedto the region.

In one method for use of this device, the collection electrode may firstcollect the materials, and then be placed repulsive relative to thecollected material, thereby sweeping the material towards the regioncontaining the array. The material may be transported in a wave mannerover the array, permitting either interaction with a passive array or anelectrically active array. Alternatively, the material may be moved overthe region of the array, and effective maintained in that position byapplication of AC fields. This embodiment has proved capable ofperformance of repeat hybridizations, where material is move to andinteracted with the array, after which it is moved out of the region,and preferably held by the collection electrode or on another electrode,after which it is moved to the array for a second, though possiblydifferent, interaction.

In yet another embodiment of device design, a substantially concentricring design is utilized. In combination, an electronic device forperforming active biological operations includes a support substrate, anarray of microlocations disposed on the substrate in a annular region, afirst counter electrode disposed on the substrate surrounding the array,and a collection electrode disposed on the substrate and disposedinterior of the array. In the preferred embodiment, the first counter orreturn electrode is segmented, optionally having pathways resulting inthe segmentation which serve as pathways for electrical connection tothe array. In yet another variation of this embodiment, multiple ringsare provided surrounding the array.

In yet another embodiment of this invention, a reduced component count,preferably five component, system is implemented in a flip-chiparrangement for providing active biological diagnostics. The devicecomprises in combination a support substrate having first and secondsurfaces and a via, pathway or hole between the first and secondsurfaces to permit fluid flow through the substrate, at least one of thefirst and second surfaces supporting electrical traces, a secondsubstrate including at least a first surface, the first surface beingadapted to be disposed in facing arrangement with at least one of thefirst and second surfaces of the first substrate and positioned near,e.g., under, the via, the second substrate including electricallyconductive traces connecting to an array of microlocations, the arraybeing adapted to receive said fluid through the via, pathway or hole,electrically conductive interconnects, e.g., bumps, interconnecting theelectrical traces on the second surface of the support substrate and theelectrical traces on the first surface of the second substrate, asealant disposed between the second face of the support substrate andthe first face of the second substrate, said sealant providing a fluidicseal by and between the first substrate and the second substrate, andoptionally, a flowcell dispose on the first surface of the firstsubstrate. Preferably, the structures utilize a flip-chip arrangement,with the diagnostic chip below the support substrate in operationalorientation. This design is particularly advantageous in reducing thenumber of components in the device, and to improve manufacturingreliability.

In yet another embodiment, an electronic device for performing activebiological operations comprises a support substrate having a first andsecond surface, and a via between the first and second surfaces topermit fluid flow through the substrate, a second substrate including atleast a first surface, the first surface being adapted to be disposed infacing arrangement with the second surface of the first substrate, thesecond substrate including an array of microlocations, the array beingadapted to receive said fluid, a sealant disposed between the secondface of the support substrate and the first face of the secondsubstrate, a source of illumination, and a waveguide having an inputadapted to receive the illumination from the source, and an outputadapted to direct the illumination to the array, the waveguide beingsubstantially parallel to the support substrate, the illumination fromthe waveguide illuminating the array. In the preferred embodiment, thesource of illumination is a laser, such as a laser bar. Such a devicemay utilize a support substrate which is flex circuit or a circuitboard.

A novel, advantageous method of manufacture may be utilized with some orall of the embodiments. The method is particularly advantageous for themanufacture of the flip-chip design. In that structure, there is a chipdisposed adjacent a substrate, the substrate including a viatherethrough, the structure being adapted to receive a fluid to beplaced on the substrate, and to flow through the via down to the chip,where at least a portion of the chip includes an area to be free ofsealant overcoat. Selection of sealant viscosity and materials mayeffectively result in effective coverage, good thermal contact betweenthe substrate and the chip, and fluidic sealing. In the most preferredembodiment, the method may include use of a light-curable sealant whichis cured with light during application. Specifically, light is exposedto the device onto the substrate and through the via, down to the chip.Next, a light curable, wickable sealant is applied to the interfacebetween the substrate and the chip. The light at least partially curesthe sealant as a result of the exposure, whereby the sealant isprecluded from flowing to said area to be free of sealant. Finally, ifdesired, the cure of the sealant may be completed, such as by heattreatment.

In yet another embodiment, a system or chip includes a multi-site arraywith electrically repetitive unit cell locations. Typically, the arrayis formed of rows and columns, most typically an equal number of rowsand columns. The individual unit cells of the array of unit cells isselected by action of selectors such as one or more row selectors andone or more column selectors. The selector may be a memory, such as ashift register memory, or a decoder, or a combination of both. An inputfor address information receives addresses, typically from off-chip,though on-chip address generators may be utilized. In the preferredembodiment, the row selectors comprise shift registers, either in a byone configuration, or in a wider configuration, such as a by fourconfiguration. In operation, the selection registers are sequentiallyloaded with values indicating the selection or non-selection of a unitcell, and optionally, the value (or indicator of value) of output forthat cell. Optionally, memory may be provided to retain those values soas to continue the output from the unit cell.

The system or chip provides for the selective provision of current andvoltage in an active biological matrix device which is adapted toreceive a conductive solution including charged biological materials. Inone aspect, an array of unit cells is provided. Each unit cell typicallyincludes a row contact and a column contact. Row lines are disposedwithin the array, the row lines being coupled to the row contacts of theunit cell. A row selector selectively provides a row select voltage tothe row lines. Further, column lines are disposed within the array, thecolumn lines being coupled to the column contacts of the array. A columnselector selectively provides a column select signal to the columnlines. The unit cells are coupled to a supply voltage and to anelectrode, the row select signal and the column select signal serving toselect a variable current output from the electrode of the unit cell. Areturn electrode is coupled to a potential and adapted to contact theconductive solution. In operation, selective activation of one or moreunit cells results in the provision of current within the conductivesolution.

In one preferred embodiment of a unit cell, a symmetric arrangement isutilized. A first column select unit, preferably a transistor, and afirst row select unit, also preferably a transistor, are in seriesrelation between a first source, e.g., voltage and/or current source,and a node, typically a current output node. In the preferredembodiment, the column select transistor may be precisely controlledunder application of a gate voltage such as from the column shiftregister memory. Preferably, the select units may differ from each otherin their controllability, such as by varying the channel length in thecontrol transistor. The channel lengths have been chosen so as to matchthe gain or other desired properties between the row and columntransistors. Also, the long channel length provides the ability tocontrol small currents with reasonable control signals. Thus, byapplication of potentials from the row selector and column selector,application of potential to the control gates results in output ofcurrent at the unit cell.

The unit cell circuit preferably further includes a second column selectunit, preferably a transistor, and a second row select unit, alsopreferably a transistor, used in series relation between a secondsource, e.g., voltage and/or current source, and a node, typically thepreviously referred to node, i.e., a current output node. In thepreferred embodiment, the first source is a supply potential Vcc and thesecond source is a reference potential, such as ground. Preferably thenodes are the same node, such that there is a series connection betweenVcc and ground of the first column select unit and first row selectunit, the node, and the second row select unit and the second columnselect unit. Optionally, the return electrode is biased at a potentialbetween the potential of the first source and the second source, e.g.,Vcc/2.

In yet another aspect of the preferred embodiment, test circuitry isincluded. Test circuitry may be utilized to ensure circuit continuity,by permitting testing prior to application of a fluidic solution. Afirst test transistor spans the first column select and first row selecttransistor. Likewise, a second test transistor spans the second columnselect and second row select transistor. Selective activation ensurescontinuity of the circuit. Alternatively, the test circuit function maybe performed by special programming of the row and column transistors,e.g., turning on of the first and second row select and first and secondcolumn select transistors.

In yet a further aspect of this invention, the current supply to thetest site is varied. Examples of the variation of current over time mayinclude static direct current (i.e., no variation as a function oftime), square wave, sinusoidal, sawtooth, or any waveform which varieswith time. In one embodiment, the currents, whether static or varying asa function of time, are supplied to the column selection circuitry,which are then selectively provided in a digital manner to the columnlines for coupling to the selected electrodes. This mixed analog anddigital technique permits significant control of the values andwaveforms of the current supplied at the individual electrodes. Thewaveforms, e.g., the current waveforms, may be generated either on-chipor off-chip. Additionally, control or operation of the overallcircuitry, and/or generation of signals such as the current waveformsmay be generated through the use of digital to analog converters (DACs),central processing units (CPUs), through the use of local memory forstorage of values, through the use of clock generators for timing andcontrol of various waveforms, and through the use of digital signalprocessors (DSPs).

In one aspect of this invention, a system based upon current control ofa first current is utilized to effect control of a second current.Preferably, a current mirror arrangement is utilized. A current supplyprovides a variable value of current for use in a voltage generationcircuit. In the preferred embodiment, multiple current sources areutilized, being summed at their output, under the selective control of amemory for selective inclusion. A variable voltage is generated at anode, preferably through use of a voltage divider circuit which receivesthe output of the variable current. The variable voltage at the node iscoupled to a control element in the unit cell, the control elementpreferably providing a variable resistance between a first voltage andan output node. The variable control element thereby provides a variablecurrent output. In this way, a first current of a relatively highervalue may be utilized to control a second current of a relativelysmaller value, the second current being supplied in operation to theconductive solution applied to the active electronic device for purposesof molecular biological analysis and diagnostics. In one embodiment, areduction of current by a factor of 32 permits provision of currents tothe device which are easily generated and controlled, yet results incurrents of a magnitude which are required for effective operation ofthe active biological device.

In yet another aspect of these inventions, the various devices may bedecorated or covered with various capture sequences. Such capturesequences may be relatively short, such as where the collectionelectrode is a complexity reduction electrode. Further, relativelylonger capture sequences may be used when further specificity orselectivity is desired. These capture sequences may preferably beincluded on the collection electrodes, or intermediate transportationelectrodes.

Accordingly, it is an object of this invention to provide an activebiological device having reduced costs of manufacture yet consistentwith achieving a small size microlocation.

It is yet another object of this invention to provide devices whichprovide increased functionality.

It is yet a further object of this invention to provide devices whichachieve a high degree of functionality and operability with fewer partsthan known to the prior art.

It is yet a further object of this invention to provide devices whichare easier to manufacture relative to the prior art.

It is yet a further object of this invention to provide circuitry andsystems which eliminate or reduce the pin limitation or pin outlimitations.

It is yet a further object of this invention to provide a system whichprovides for precise current control in an active electronic deviceadapted for molecular biological analysis and diagnostics, which mayinterface with larger currents generated by a control system.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show an active, programmable electronic matrix device(APEX) in cross-section (FIG. 1A) and in perspective view (FIG. 1B).

FIG. 2 is a plan view of an embodiment of the invention which utilizesvarying sized electrode regions and focusing electrodes, variouslyreferred to as the bug chip.

FIG. 3 is a plan view of an embodiment of the invention which utilizes aconcentration electrode and paired return electrode, which is especiallyuseful in methods which effectively transport charged biologicalmaterial in a wave or sweeping motion across microlocations.

FIG. 4 is a plan view of an embodiment of the invention which utilizes asubstantially circular arrangement, with a substantially centrallydisposed concentration electrode.

FIGS. 5A, 5B and 5C show perspective views and FIG. 5D shows across-sectional view of a flip-chip system, FIG. 5A showing theunderside of the system, FIG. 5B showing a perspective view of top ofthe flip-chip structure including sample chamber, FIG. 5C showing a topperspective detail of the via, and FIG. 5D showing a cross-sectionalview of the flowcell.

FIGS. 6A and 6B show perspective and cross-sectional views,respectively, of a flip-chip system in one embodiment.

FIGS. 7A and 7B shows side and plan views, respectively, of an edgeilluminated system in one embodiment of this invention.

FIG. 8 is a microphotograph of barrier wall for the Norland 83H damusing a 1300 J/s fiber bundle source shadow masked with the flex circuit(Flex polyimide removed).

FIG. 9 is a block diagram drawing of a multiple unit cell array system.

FIG. 10A is a circuit diagram of a functionalized unit cell usable withthe system of FIG. 9.

FIG. 10B is a voltage/timing diagram for the circuit of FIGS. 9 and 10A.

FIG. 10C are current diagrams as a function of time for the circuit ofFIGS. 9 and 10A.

FIG. 11 is a component level circuit diagram of a unit cell usable withthe system of FIG. 9.

FIG. 12 is a component level circuit diagram of a unit cell includingadditional test circuitry usable with the system of FIG. 9.

FIG. 13 is a schematic diagram of a circuit for providing currentcontrol in an active electronic device.

FIG. 14 is a component level circuit diagram of current mirrors.

FIG. 15 is a component level circuit diagram of column selectioncircuitry.

FIG. 16 is a component level schematic diagram for a row select circuit.

FIG. 17 is a plan view of a physical layout of a unit cell.

FIG. 18 is a plan view of a layout of a portion of the 20×20 test siteunit.

FIG. 19 is a block diagrammatic view of the overall control and testsystem in one aspect of this invention.

FIG. 20 is a schematic block diagram view of the interconnection betweenan input system and a probe card for connection to an active biologicalmatrix system.

FIG. 21 is a graph of hybridization as a function of specific andnon-specific hybridization for field-shaping and for no use of fieldshaping.

FIG. 22 is a graph of Average MFI/s at various concentrations for theembodiment of FIG. 2, at various concentrations RCA5 BTR Reporter in 50mM histidine, showing Specific/Non-Specific Binding After Washing.

FIG. 23 is a graph of current linearity showing the electrode currentoutput in nanoamps as a function of current n in microamps.

DETAILED DESCRIPTION OF THE INVENTION

FIGS. 1A and 1B illustrate a simplified version of the activeprogrammable electronic matrix (APEX) hybridization system for use withthis invention. FIG. 1B is a perspective view, and FIG. 1A is across-sectional view taken in FIG. 1B at cut A-A′. Generally, asubstrate 10 supports a matrix or array of electronically addressablemicrolocations 12. For ease of explanation, the various microlocationshave been labeled 12A, 12B, 12C and 12D. A permeation layer 14 isdisposed above the individual electrodes 12. The permeation layerpermits transport of relatively small charged entities through it, butreduces or limits the mobility of large charged entities, such as DNA,to preferably keep the large charged entities from easily contacting theelectrodes 12 directly during the duration of the test. The permeationlayer 14 reduces the electrochemical degradation which would occur inthe DNA by direct contact with the electrodes 12, possibly due, in part,to extreme pH resulting from the electrolytic reaction. It furtherserves to minimize the strong, non-specific adsorption of DNA toelectrodes. Attachment regions 16 are disposed upon the permeation layer14 and provide for specific binding sites for target materials. Theattachment regions 16 have been labeled 16A, 16B, 16C and 16D tocorrespond with the identification of the electrodes 12A-D,respectively. The attachment regions 16 may be effectively incorporatedinto or integrated with the permeation layers (e.g., 12A), such as byincluding attachment material directly within the permeation material.

In operation, reservoir 18 comprises that space above the attachmentregions 16 that contains the desired, as well as undesired, materialsfor detection, analysis or use. Charged entities 20, such as charged DNAare located within the reservoir 18. In one aspect of this invention,the active, programmable, matrix system comprises a method fortransporting the charged material 20 to any of the specificmicrolocations 12. When activated, a microlocation 12 generates the freefield electrophoretic transport of any charged functionalized specificbinding entity 20 towards the electrode 12. For example, if theelectrode 12A were made positive and the electrode 12D negative,electrophoretic lines of force 22 would run between the electrodes 12Aand 12D. The lines of electrophoretic force 22 cause transport ofcharged binding entities 20 that have a net negative charge toward thepositive electrode 12A. Charged materials 20 having a net positivecharge move under the electrophoretic force toward the negativelycharged electrode 12D. When the net negatively charged binding entity 20that has been functionalized contacts the attachment layer 16A as aresult of its movement under the electrophoretic force, thefunctionalized specific binding entity 20 becomes covalently attached tothe attachment layer 16A. Optionally, electrodes 24 may be disposedoutside of the array. The electrodes 24 may optionally serve as returnelectrodes, counter electrodes, disposal (dump) electrodes or otherwise.Optionally, a flowcell may be provided adjacent the device for fluidiccontainment.

FIG. 2 is a plan view of one embodiment of the invention which utilizesfocusing electrodes 42, 44, and optionally, transport electrodes 50, 52,54. The device 20 includes a substrate 32, which may be of anysufficiently rigid, substantially non-conductive material to support thecomponents formed thereon. The substrate 32 may be flex circuit (e.g., apolyimide such as DuPont Kapton, polyester, ABS or other suchmaterials), a printed circuit board or a semiconductive material,preferably with an insulative overcoating. Connectors 34 couple totraces 36, which in turn, couple to other electrical components of thesystem. These components may be any form of conductor, such as copper,or gold, or any other conductor known to those skilled in the art.Various of the connectors 34 are shown unconnected to traces 36 or otherelectrical components. It will be appreciated by those skilled in theart that not every connector 34, such as in a system adapted to matewith an edge connector system will be utliized. Additionally, traces 36may be of differing widths depending upon the demands, especially thecurrent demands, to be made on that trace 36. Thus, some traces 36 maybe wider, such as those being coupled to the focusing electrodes 42, 44,in comparison to those traces 36 coupled to the microlocations withinthe array 38. Array 38 is preferably of the form described in connectionwith FIGS. 1A and 1B.

A first collection electrode 40 and counter electrodes 46 are disposedon the substrate 32. These components generally fit within the footprint(shown in dashed line) of the flow cell 58, and comprise a relativelylarge percentage thereof, preferably at least substantially 40%, andmore preferably substantially 50%, and most preferably substantially60%. The counter electrodes 46 (sometimes functioning as returnelectrodes) and collection electrode 40 are preferably disposed at ornear the periphery of the flow cell footprint 58, and may substantiallycircumscribe, e.g., to 80%, the footprint perimeter.

Typically, the collection electrode 40 and counter electrodes 46 aredisposed on the substrate 32 so that the electrophoretic lines of forceare significant over substantially all, e.g., 80% or more, of the flowcell volume. By way of example, the concentration 40 and counterelectrodes 46 may be disposed near the periphery of the footprint 58 ofthe flow cell. In yet another embodiment, they may be disposed atsubstantially opposite ends of the flowcell footprint 58 (See, e.g.,FIG. 3). In yet another embodiment, the counterelectrode substantiallycircumscribes the footprint of the flow, with a centrally disposedcollection electrode (See, e.g., FIG. 4). The relatively large percentof coverage of the flow cell footprint 58 and its position aids ineffective electrophoretic interrogation of the flow cell contents.

Returning to FIG. 2, focusing electrodes 42, 44 are disposed on thesubstrate 32 to aid in focusing materials collected on the collectionelectrode 40 to the array 38. The focusing electrodes 42, 44 arepreferably disposed in a mirror-image, “Y” or “V” shaped pattern, theopen end encompassing, at least in part, the collection electrode 40. Asshown, there are two symmetric focusing electrodes 42, 44. One focusingelectrode may be utilized, or more than two focusing electrodes may beutilized. As shown, the focusing electrodes 42, 44 include substantiallyparallel portions (adjacent the array) and angled portions (adjacent thetransport electrodes 50, 52, 54, and optionally, the collectionelectrode 40) extending in a symmetrical manner enveloping the transportelectrodes 50, 52, 54. Stated otherwise, there are first and secondelectrodes being disposed at least in part adjacent the array ofmicrolocations, the distance between the first and second electrodesadjacent the array being smaller than the distance between the first andsecond electrodes in yet another region disposed away from the array.The focusing electrodes 42, 44 may optionally include portions which aredisposed on the opposite side of the array 38 from the collectionelectrode 40. The focusing electrodes 42, 44 are preferably coupled toleads 36 which are relatively larger than the leads 36 coupled to thearray 38, so as to permit the carrying of effective currents andpotentials.

Transport electrodes 50, 52, 54 are optionally included. Electrodes ofmonotonically decreasing size as they approach the array 38 are shown. Afirst transport electrode 50 is relatively smaller than the collectionelectrode 40, the second transport electrode 52 is relatively smallerthan the first transport electrode 50, and the third transport electrode54 is yet smaller still. The differential sizing serves to reducecurrent density mismatches between locations, and aids in reducing oreliminating burn-out which may result if too great a current densitymismatch exists. Transport efficiently is maximized. The ratio of sizesof larger to smaller is preferably substantially 2 to 1, more preferably3 to 1, and may be even greater, such as 4 to 1 or higher.

One field-shaping protocol is as follows:

Negative Bias Positive Bias Current Bias Time Counter Electrodes 46 1stCollection Electrode 40 75 μA  30 sec. Focusing Electrodes 42, 44 (−0.2μA) 1st Transport Electrode 50 25 μA  90 sec. 1st Collection Electrode40 Focusing Electrodes 42, 44 (−0.2 μA) 2nd Transport Electrode 52 5 μA180 sec. 1st Transport Electrode 50 Focusing Electrodes 42, 44 (−0.2 μA)3rd Transport Electrode 54 3 μA 420 sec. 1st Transport Electrode 50 2ndTransport Electrode 52 Focusing Electrodes 42, 44 (−0.2 μA) Row 3 1.5 μA120 sec. 2nd Transport Electrode 52 (500 nA/pad) 3rd Transport Electrode54 Focusing Electrodes 42, 44 (−0.2 μA) Row 2 1.5 μA 120 sec. 2ndTransport Electrode 52 (500 nA/pad) 3rd Transport Electrode 54 FocusingElectrodes 42, 44 (−0.2 μA) Row 1 1.5 μA 120 sec. 2nd TransportElectrode 52 (500 nA/pad) 3rd Transport Electrode 54

The seven steps of the field shaping protocol serve to effectivelyinterrogate the sample volume and to correct materials onto the array 38for analysis. In the first step, interrogation of the sample volume iseffected through negative bias of the counter electrodes 46 and positivebias of the first collection electrode 40. The placement of thecounterelectrodes 46 and collection electrode 40 generally near theperiphery of the footprint of the flow cell 58 permit the rapid,effective interrogation of that sample volume. Secondly, with thecollected material adjacent the collection 40, that electrode is madenegative (repulsive) to materials of interest, while the first transportelectrode 50 is made positive (attractive). The repulsion and attractioneffects transport of materials from the collection electrode 40 to thefirst transport electrode 50. Additionally, the focusing electrodes 42,44 are biased negative. Such a negative (repulsive) bias serves toprovide a force which may be lateral to the direction of transport,thereby more centrally concentrating material in the solution. Thirdly,with material collected at the first transport electrode 50, thatelectrode may be biased negative (repulsive), while the second transportelectrode 52 is biased positive (attractive). The focusing electrodes42, 44 may be biased negatively, which serves to provide a repulsiveforce on the charged materials, thereby providing a transverse componentto their direction of motion and collecting the material within asmaller physical region or volume. Fourth, the second transportelectrode 52 may be biased negative, as well as optionally biasing ofthe first transport electrode 50, to effect transport away from thoseelectrodes and to the now positively biased third transport electrode54. Again, the focusing electrodes 42, 44 may retain their negativebias. The next three steps are optionally separated, as described, totransport materials to various rows or regions of the array 38.

The field shaping protocol includes currents and biased times. In thisembodiment, there is an inversely proportional relationship between thesize of the electrode and the amount of current supplied to it. Further,for the collection electrode 40 and transport electrodes 50, 52 and 54,there is an inversely proportional relationship between the electrodesize and the bias time, that is, the smaller the electrode, the largerthe bias time. Through this protocol, the current density at variousdevices is kept relatively more uniform, optionally substantiallysimilar to each other. Further, as the current from a given electrodedecreases (relative to a larger electrode) a relatively longer bias timemay be required in order to provide transport of effective amounts ofcharged material between the various electrodes. Stated otherwise, for agiven amount of charged material, a relatively longer bias time may berequired to effect transport of a given amount of material at a lowercurrent.

FIG. 3 is a plan view of another embodiment of this invention. As withFIG. 2, a device 60 includes a substrate 62, connectors 64, traces 66and an array of microlocations 68. The comments made for FIG. 2 andothers apply to corresponding structures in other figures. Further, thetraces 66 leading from the upper left portion of the array 68 have beentruncated for drawing simplicity. A corresponding arrangement to thoseshown in the lower right of the drawing would apply. The traces 66 maybe of the same width or of varying width, such as where a relativelywider trace 66 may be utilized for larger current carrying capacity(e.g., traces 66 to first collection electrode 70 and second collectionelectrode 72.

FIG. 3 departs from FIG. 2 in the inclusion of a first collectionelectrode 70, being disposed at least in part adjacent the array 68. Inthe embodiment of FIG. 3, first collection electrode 70 is a trapezoid,which has a long base 70 b adjacent to and parallel to one side of thearray 68 and top 70 t, which is preferably shorter than the base 70 b,with sloping sides 70 s, tapering wider (away from each other) towardthe base 70 b. The second collection electrode 72 is disposed on theother side of the array 68, and is similarly (though not necessarilyidentically) shaped and sized. Top 72 t is preferably shorter than base72 b, and accordingly, the sides 72 s are non-parallel and slope awayfrom each other, moving towards the array 68. Optionally, the electrodes70, 72 may be of different sizes, such as where the area of the firstcollection electrode 70 is approximately 10% smaller (optionallyapproximately 20% smaller) than the second collection electrode 72.Input port electrode 74 and port electrode 76 are optionally included onthe substrate 62, within the footprint of the flow cell 78. The inputport electrodes 74 and port electrode 76 are either of the same size orof different size.

In operation, the flow cell contents are interrogated by placing orbiasing one of the first and second collection electrodes 70, 72attractive (typically positive) to the materials to be collected. Oncecollected, the materials may be transported away from the firstcollection electrode 70 towards the array 68. The materials may beeffectively held in place over the array 68, such as by application ofAC fields such as at a frequency in the range from 0.01 to 10⁶ Hz, mostpreferably between 0.1 to 10³ Hz between the electrodes 70, 72. Thenmaterials may be transported to the other electrode 70, 72 or may berepeatedly reacted by moving materials from the array 68 to theelectrodes 70, 72. Optionally, the microlocations of the array 68 may beelectrically active or passive.

FIG. 4 is a plan view of a concentric ring electrode embodiment. Thedevice 80, substrate 82, connectors 84, traces 86 and array 88 are aspreviously described, with the exception that the array 88 may bearranged concentrically. A concentric return electrode 90 and centralconcentration electrode 92, preferably round, coact to concentratematerial at electrode 92, and then to move it over or position it abovethe array 92. As with the preceding FIGS. 2 and 3, the traces have beenshown in a truncated manner.

In the embodiments of FIGS. 2, 3 and 4, capture sequences or probes maybe disposed on the devices. Preferably these are at least on thecollection or concentration electrodes. Optionally, different sequencesare disposed on different devices such as the transport electrode 50, 52and 54 of FIG. 2. For example, each sequence as an approach is made tothe array may be more specific.

FIGS. 5A, 5B, 5C and 5D show views of the bottom, the top, the top withvia 109 exposed, and a side view of the system through cut A-A′ in FIG.5B, respectively, of a flip-chip system. A device 100 includes a supportsubstrate 102 having a first surface 104 (optionally called the topsurface) and a second surface 106 (optionally called the bottomsurface), which may be of materials suitable for the function of supportand conduction, such as flex circuitry, printed circuit board,semiconductive material or like material. Contacts 108 lead to traces10, which lead to the second substrate 112. This second substrate 112may also be referred to as the flipped chip. This second substrate 112may optionally be a chip, system or support on which assays or otherdiagnostic materials are provided. Contacts, such as bump contacts,e.g., solder bumps, indium solder bumps, conductive polymers, or silverfilled epoxy, provide electrical contact between traces 110 and the chipor substrate 112. A sealant is disposed between the second (bottom)surface 106 of the support substrate 102 and the first (top) surface 114of the second substrate 112. Generally, the opposing faces of thesupport substrate 102 and second substrate 112 are those which areplaced in fluid-blocking contact via the inclusion of a sealant. Aninlet port 120 may be in conductive relation to a sample chamber 122,which yet further leads to the assay chamber 124, and on to the outletport 126. FIG. 5C shows a perspective view of the support 102 and thevia 128 formed through it. As shown, the lateral width of the via 128 isless than the lateral width of the second substrate 112. The secondsubstrate 112 is shown in dashed lines, which is disposed below thesubstrate 102 in the view of FIG. 5C.

In the preferred embodiment, the device 100 is formed of a minimumnumber of components to reduce cost, improve manufacturing simplicityand reliability or the like. One embodiment is achieved in substantiallyfive components. While the device may be fabricated with fivecomponents, the addition of components which do not detract from or varythe inventive concept may be utilized. These components are as follows.First a support substrate 102 having a first surface 104 and secondsurface 106, and avia 128 between the first surface 104 and secondsurface 106 to permit fluid flow through the substrate 102, the secondsurface 106 supporting electrical traces. Second, a second substrate 112including at least a first surface 114, the first surface being adaptedto be disposed in facing arrangement with the second surface 106 of thefirst substrate, the second substrate 112 including electricallyconductive traces connecting to an array of microlocations (See, FIG. 1Aand 1B), the array being adapted to receive said fluid through the via128. Third, electrically conductive bumps 128 interconnecting theelectrical traces on the second surface of the support substrate and theelectrical traces on the first surface 106 of the second substrate.Fourth, a sealant 130 disposed between the second face 106 of thesupport substrate 102 and the first face 114 of the second substrate112, said sealant 130 providing a fluidic seal by and between the firstsubstrate 102 and the second substrate 112. Fifth, a flowcell isoptionally disposed on the first surface 104 of the first substrate 102.While the number of elements may vary, advantages may be obtained fromselection of these five elements.

In operation, a sample is provided to the inlet port 120 and passed tothe sample chamber 122. The sample chamber 122 may serve to housevarious sample processing functions, including but not limited to cellseparation, cell lysing, cell component separation, complexityreduction, amplification (e.g., PCR, LCR, enzymatic techniques), and/ordenaturation). Thereafter, the sample flows to the assay chamber 124.Solution containing sample flows down through via 128 (which is obscuredin FIG. 5B by assay chamber 124, though may be seen in FIG. 5C and 5D).A space is formed comprising the via 128, bounded on the bottom by thesecond substrate 112, with sealant or adhesive 130 forming a barrierbetween the interface of the second surface 106 of the support substrate102 and the first surface 114 of second substrate 112.

In the preferred method of manufacture, a light curable sealant iswicked or otherwise provided to the interface between the second surface106 of the support substrate 102 and the first surface 114 of the secondsubstrate. Light is provided through the via 128. A dam is formed,stopping the advance of the sealant, thereby maintaining the array,e.g., 18, substantially free from sealant or adhesive. (See FIG. 8 for amicrophotograph showing the sealant free area of the array, the curedleading edge of the dam and sealant on the exterior portions of thedevice.) By the appropriate sizing of the lateral width of the via 128,the via 128 serves essentially as a shadow mask for the incident light,which serves to cure the sealant. Alternatively, the sealant may besupplied to the interface between the second surface 106 of the supportsubstrate 102 and the first surface 114 of the second substrate 112 inan amount and with a viscosity such that it does not flow onto the array18. Further or final curing of the sealant may be performed as required,such as by heating.

FIG. 6A shows a perspective exploded drawing and FIG. 6B shows across-sectional view, respectively, of a flip-chip system in accordancewith one implementation of this invention. The system of FIGS. 6A and 6Binclude an edge illumination member 140, unique sample chamber 134design, and a ‘butterfly’ input and output chamber design as compared toFIGS. 5A, 5B and 5C. A chip or substrate 130 has a first surface 130 tand a second surface 130 b, at least the first surface 130 t includingelectrical regions or traces 132 thereon or therein. While theembodiment shown in cross-section in FIG. 6B shows the trace 132disposed on the top surface 132 of the chip or substrate 130, theelectrical regions may be contained wholly or partially within the chipor substrate 130, such as through the provision of semiconductiveregions. These semiconductive regions may be controlled in an activemanner so as to provide selective connections within the chip orsubstrate 130. Typically, the first surface 130 t is that surface onwhich the active biological interactions will take place. Optionally, anedge illumination member 140 may be disposed adjacent to andsubstantially coplanar with the first surface 130 t of the chip orsubstrate 130. The illumination sheet 140 preferably includes holes,vias or pathways 144 to permit electrical interconnections 156 to passtherethrough. As can be seen, the illumination sheet 140 may be disposeddirectly over conductive traces 132 or may be directly affixed to theadjacent supporting sheet 150. Electrical traces 132 may be included onthe first surface 130 t of the substrate or chip 130. An electricallyconductive element 136, such as a solder connection, indium bump,conductive polymer or the like couples the conductive pathway 132 on thesubstrate 130 to the conductive portion of the contact trace 154. Thecontact trace preferably is then contacted by a conductive member 156,such as a wire, whisker wire, or other electrical contact, forconnection to the remainder of the circuit. Sealant 180 is preferablydisposed between the substrate or chip 130 and the next layer 150, suchas the flex support layer.

In FIG. 6B, the drawing has been presented with a conductive member 136on the left hand side, but with sealant 180 on the right hand side. Itwill be appreciated that other conductive members 136, not disposed inthe plane of the cut, are included and provide further mechanicalsupport between the substrate 130 and the trace support layer 150.Further, the edge illumination layer 140 includes a terminal edge 142,which is disposed toward the upper surface 130 t of the substrate orchip 130. The edge illumination layer 140 may terminate outside of orinside of the sealant 180. An adhesive layer 160 is disposed adjacentthe trace support layer 150, and provides adhesive contact to an upperlayer 170. The upper layer 170 may optionally include pathways,indentations, or other cutouts, such as shown for an inlet 176 and anoutlet 176′. As shown, the adhesive layer 160 may optionally be adie-cutable adhesive material, such as one which includes release paperon both the top surface and the bottom surface prior to assembly.Suppliers of such materials include 3M or Dupont. As shown in FIG. 6A,the die-cutable adhesive material 160 may be cut so as to form all or apart of the wall 162, 164 of a chamber. As shown, the geometry may bemade in any desired shape or flow cell configuration.

Preferably, a top member 170 is provided. As shown, the top member 170may extend substantially over the remainder of the device. Optionally,the top member 170 may form a window 172 or other containment surface atthe top of the flow cell chamber. Preferably, the top material is formedfrom polycarbonate or polystyrene. In the configuration of FIGS. 6A and6B, it is typically contemplated that the array of test sites will beaccessed optically through the top member 172, and accordingly, it isdesirable to form the top member from materials which are substantiallytransparent to both the excitation and emission radiation.

FIGS. 6A and 6B show one geometry for a flow cell. In this ‘butterfly’configuration, the inlet 176 is connected to a first expanding region174 wherein the sidewalls of the chamber start at a first dimension dand expand, preferably monotonically, and most preferably linearly, to adimension D at a point closer to the flow cell chamber 134. The flowcell chamber 134 region is characterized by substantially parallelsidewalls 166. Preferably, a first decreasing width region is providedbetween the flow cell region and the output. Most preferably, thedecreasing region begins with a width D′, most preferably where D′=D,and decreases to a width d′, preferably where d′=d. As can be seen inFIGS. 6A and 6B, the height of the inlet chamber 174 decreases from theinlet at a height H to a lesser height h at the inlet to the flow cellchamber. Preferably, the decrease is monotonic, and most preferably,linear.

In the preferred embodiment, the height h of the inlet chamber and thewidth w are chosen such that a substantially constant flow area isprovided, that is, the product of the height h and the width w (h×w) issubstantially constant. Thus, as shown in the combined view of FIGS. 6Aand 6B, at the portion of the inlet chamber adjacent the inlet, whilethe height h is relative large, the width w is relatively small.Correspondingly, when proceeding through the inlet chamber towards theflow cell chamber, as the width w increases, the height h decreases.Preferably, the outlet chamber includes substantially the same geometry,and preferably the same flow cell area constant.

FIGS. 7A and 7B are cross-sectional and plan views, respectively, of anedge illuminated, flip-chip system in accordance with one embodiment ofthe invention. To the extent possible, a consistent numbering ofelements from FIGS. 6A and 6B will be utilized. A support substrate 150is generally planar, and includes a first face 150 t and a second face150 b. A via 126 (shown in dashed lines for the cross-section) permitsfluid or solution flow from above the support substrate 150 to thesecond substrate 130, particularly to the first surface 130 t of thesecond substrate 130. Sealant 180 is provided between the second face150 b of the support substrate 150 and the second substrate 130. Thesealant 180 provides a preferably fluid tight-seal, so as to permitfluid flow to the array on the second substrate 130. A source ofillumination 190, such as a laser bar, illuminate the array on thesecond substrate 130. Preferably, the system includes a waveguide 140with an input 146 adapted to receive illumination from the source 190,and to provide illumination via output 142. The waveguide 140 ispreferably co-planar with the support substrate 150, and may be securedto it, such as by being adhered to the second surface 150 b of thesupport substrate 150. Electronics 192 may be included to control thesystem. Optionally, surface mounted electronic components may beincluded on the substrates 130, 150. Fluidics 194 may be provided incombination with the system to aid in provision of the sample to thesecond substrate 130.

FIG. 9 is a block diagrammatic depiction of a multiple unit cell array.In the preferred embodiment, a system or chip includes a multi-sitearray 210 with electrically repetitive site cell locations. Typically,the array is formed of rows and columns, more typically an equal numberof rows and columns, yet most typically in an orthogonal arrangement forrows and columns. For example, an array of 10×10, 20×20 or more may beformed with these techniques. The individual unit cell 212 of the array210 of unit cells is selected by action of selectors such as a rowselector 220 and a column selector 230. The selectors 220, 230 may be amemory, such as a shift register memory, or a decoder, or a combinationof both. An input for address information receives addresses, typicallyfrom off-chip, though on chip address generators may be utilized. In thepreferred embodiment, the row selectors 220 comprise shift registers,either in a by one configuration (×1), or in a wider configuration, suchas a by four configuration (×4). In operation, the selection registersare sequentially loaded with values indicating selection, or not, of aunit cell 212, and optionally, the value of output for that cell.Optionally, memory may be provided to retain those values so as tocontinue the output from the unit cell.

Considering FIG. 9 in more detail, an array 210 includes a plurality ofunit cells 212. In the preferred embodiment, the unit cells 212 arearranged in rows and columns, the designation row in FIG. 9 depicting ahorizontal arrangement relative to the text, and a column designating avertical arrangement relative to the text (though it will be understoodby those skilled in the art that the designations row and column may bereversed). The designation row or column may also refer to a group orsubset of unit cells 212, such as a portion of a row or column, or agroup or set of unit cells 212 which are not linearly contiguous. Ingeneral, there are m rows and n columns of unit cells 212, typicallywhere m=n, and m=2, 3, 4 . . . By way of example, a 5×5 matrix of unitcells 212, a 10×10 matrix of unit cells 212 and a 20×20 matrix of unitcells 212 provides for a total number of unit cells of 25, 100 and 400,respectively.

In FIG. 9, various levels of complexity of unit cell 212 are shown. Theuppermost depicted unit cell 212 is depicted as a single block-diagramunit, whereas the unit cell 212 disposed central to the figure is shownin greater complexity, akin to the structure disclosed and described inmore detail in FIG. 10. It will be understood that these alternativesare depicted for expository convenience and variety, and that in atypical implementation, the construction of the individual unit cells212 will be the same for a given device.

The unit cells 212 are addressed by action of at least one row selector220 and at least one column selector 230. This detailed descriptionbegins with the case of a single row selector 220 and column selector230, and later describes the use of additional selectors 220′, 230′. Rowselector 220 receives input information 222 and outputs a row selectionsignal 294 (see FIG. 10A) on one or more row lines 224. The selectionsignal on the row line 224 is supplied to the unit cell 212, andinteracts therewith such as through a row contact 226. As drawn, aportion of row line 224 is shown disposed to the left and a portionshown drawn to the right of the unit cell 212 centrally disposed in thearray 210. In typical implementation, the row line 224 will beelectrically continuous, though may be made of any combination ofmaterials. For example, the row line may be one continuous conductiveline, such as formed of conductive polysilicon, or may be a combinationstructure such as where conductive segments are electrically connectedvia a higher conductivity material, such as metal, such as aluminum.

The column selector 230 receives an input 232 for determining theselection of a column, or in the preferred embodiment, the value (orcorrelated value) of the output at the unit cell 212. The columnselector 230 is coupled to the column lines 234 which serves to providea column select signal 296 a-d to the unit cells 212. In the preferredembodiment, the column selector 230 selects more than two states (e.g.,four states 296 a-296 d), preferably voltage states, which are suppliedvia the column line 234 to the unit cell 212. The column line 234 iscoupled to the unit cell 212, such as through a column contact 236. Inthe preferred embodiment, the column contact may be a control gate for atransistor, such as a field effect transistor. (See, e.g., FIGS. 11 and12).

If required for activation of the unit cell 212, a second row selector220′, input lines 222′, second row lines 224′ and column contacts 226′may be included. Likewise, a second column selector 230′ may be added,having an input 232′, and being coupled to secondary or supplementalcolumn lines 234′ which in turn are coupled to secondary column contacts236′.

As shown, the row selectors 220, 220′ and column selectors 230, 230′optionally include an enable input 228, 228′ or chip select 238, 238′.One of the functions of these signals is to permit entry of inputinformation 222, 222′, 232, 232′ without the activation of a row line224, 224′, or column line 234, 234′. Further, some or all of the rowselectors 220, 220′ and column selectors 220, 220′ and column selectors230, 230′ may include an output 229, 229′, 239, 239′ which may be usedfor output of information. In one application, the output value may be asignal or bit, such as the most significant bit of a series, indicatingthat the input data has been successfully loaded into the row selector,220, 220′ or column selector 230, 230′. Optionally, this outputinformation may be utilized to then trigger the enable or chip selectsignals 228, 228′, 238, 238′.

Within the level of detail of FIG. 9, the row selectors 220, 220′ andcolumn selectors 230, 230′ function to receive row and column inputinformation 222, 222′, 232, 232′ and to use that to select one or moreunit cells 212, and optionally, to provide signal values indicative ofthe level of current (potential) to be provided from the unit cell 212.The selectors 220, 220′, 230, 230′ may be in the form of memory, such asin the form of a shift register memory (See FIGS. 15 and 16 for detail),or may be in the form of a decoder circuit, such as where the desiredaddresses are provided as input information and the output is then in adecoded relationship thereto. Numerous circuits are known to thoseskilled in the art to effect this functionality.

A current source 240, such as a current mirror, optionally receives asource of current 242 and a control signal 244 (VCASP). Connections 246,246′ couple the current from the source 240 to column selector 230, andif present, second column selector 230′. As shown, the coupling lines246, 246′ are separate wires (designated “a” to designate a number ofwires equal to a). Further, one or more sources of current 242 may besupplied. As will be described in connection with FIG. 10C, below, thevalue of the current may be static, or may vary over time (such as inthe application of a pulsed waveform, sinusoidal waveform, square wave,sawtooth, etc.). Generally, any desired varying waveform may beutilized.

Utilizing the structure shown in FIG. 9, each of the unit cells 212 maybe activated at a given time. Alternatively, certain unit cells 212 maybe activated and yet other unit cells remain inactive. By way ofexample, if a given column has been selected at a first value, each ofthe unit cells within that column which are associated with one or moreselected rows selected by the row selector 220 will be activated at thevalue corresponding to that level of the voltage on the column. Yetother unit cells within that same column may be placed at the same or adifferent level by coupling to the second column selector 230′, wherethe one or more row lines associated with those unit cells are driven bythe second row selector 220′. Thus, within one column of unit cells,each unit cell 212 may be either driven at a value corresponding to thesignal on the column 234 associated with the column selector 230, orwith the value on the column associated with the second column selector230′, or be in an undriven, unconnected, floating or high impedancestate. In a like manner, other columns may be set to desired levels ofoutput. In this way, the entire array of unit cells may be placed in thedesired state or set of states. The use of the terms ‘levels of output’and ‘desired state’ include signals which vary as a function of time.Additionally, more values within a given column 234, 234′ may be added,such as through the addition of further column selectors and columnlines which are coupled to selected unit cells 212.

FIG. 10A shows a schematic block diagram of a unit cell 212 and returnelectrode 250. To the extent possible, the numbering convention in FIG.10 corresponds to that adopted in FIG. 9. A variable current controlelement 260 includes an input 262, an output 264 and a control element266. The control element 266 is coupled to a line, such as the columnline 234, which is in turn coupled to the column select 230. A selectorswitch 270 includes an input 272, an output 274 and a control element276. The control element 276 is coupled to a control line, such as a rowline 224. The output 264 of the variable current control element 260 iscoupled to the input 272 of the select switch 270. The output 274 of theselect switch 270 couples to a node 280 which provides the outputcurrent 282, I_(out). A first potential 284, e.g., Vcc, is provided toinput 262 of the variable current control element 260.

In operation, application of a signal on row line 224 to the input 276of the select switch 270 provides a conductive path between node 280 andoutput 264 of the variable current control element 260. The signal valueapplied to the column line 234, which is coupled to the input 266 of thevariable current control element 260 serves to provide a variable amountof current flowing through the series connected variable current controlelement 260 and select switch 270 between the first potential node 284and the output node 280. A return electrode 250 serves to complete thecircuit, though it will be appreciated that the return electrode 250 maybe yet another unit cell 212.

In the preferred embodiment, the variable current control element 260 isa transistor, such as a field effect transistor, and most particularly aMOSFET. The select switch 270 is preferably a transistor, morepreferably a field effect transistor, and most particularly a MOSFET.Various types of particular implementation may be utilized, whetherC-MOS, N-MOS, CMOS, bipolar, gallium arsenide, or otherwise, so long asconsistent with the functional requirements of the system. Further, inthe preferred embodiment, a matching arrangement of a second variablecurrent control element 260′ and second select switch 270′ couplesbetween a second potential 284′ and the output node 280. Optionally,channel lengths of the various devices may be arranged such that asymmetric arrangement is implemented. For example, in a CMOSimplementation, the p-channel select device may have a shorter channellength than the n-channel device, to compensate for the differingelectron/hole mobility. (e.g., 80 μ v. 126μ channel length). A similarnumbering s primes. The discussion regarding the circuit, above, appliesto the circuitry including the second variable current control element260′ and second select switch 270′.

FIG. 10B shows signals as a function of time for exemplary controlsignal of the unit cell 212. The generation of the output signal 290indicates the completion of the entry of the data into the selectors220, 220′, 230, 230′. The output signal 290 may then be used to triggeror activate the enable signal 292. The enable signal 292 in turn maypermit the select signals for the row select 294 and column selectsignals 296A, 296B, 296C and 296D pass to the unit cell 212. Asdepicted, a single row select signal 294 is provided, wherein thatsignal is provided to a select circuit 270, 270′ having preferablybistate operation. The column select signals 296A, 296B, 296C and 296Dmay be of differing values, preferably of more than two values, in thepreferred embodiment comprising at least four values, which are thenprovided to the inputs 266, 266′ of the variable current controlelements 260, 260′. As explained further, below, these values may bestatic or dynamic.

FIG. 10C depicts exemplary values of current (or voltage) as a functionof time which may be supplied to the electrodes. In one embodiment, astatic, direct current (sourced or sinked), which does not vary as afunction of time may be supplied. While the value of the current isstatic, it will be understood that the selection, typically digitalselection, of whether to permit this current to drive the electrode ornot is utilized, such that the electrode is selectively driven as afunction of time. The second waveform in FIG. 10C shows a square wave.The square wave may be for unit directional current or bidirectionalcurrent. An offset bias may be utilized as desired. As shown in thethird waveform in FIG. 10C, the waveform may have a periodicity whichhas a subcomponent waveform included within it. The fourth waveform inFIG. 10C shows a generally sinusoidal waveform. The fifth waveform inFIG. 10C shows a sawtooth waveform. It will be appreciated that anywaveform consistent with the goals and objects of this invention may beutilized in conjunction with the devices and methods disclosed herein.By supplying a waveform, most particularly, a current waveform, which isselectively controllable by digital selection (such as through theaction of the row selector 220 in FIG. 9), a high degree of flexibilityand control is achievable. Further, the waveforms supplied to varioustest sites within the device need not be the same. For example, thefirst column may have a static, direct current waveform applied to it,the second column may have a square wave waveform applied to it, whereasthe third column has a sinusoidal waveform applied to the microlocationsin that column as selected by the row selector, and so on.

The waveforms for the current may be generated on the chip or off chip.In practical implementation, the waveforms may be generated through theuse of digital to analog converters (DACs), digital signal processors(DSPs), variable current waveform generators, on-chip memories, alloptionally under control of a control system utilizing a centralprocessing unit (CPU) or other version of microprocessor control.

FIGS. 11 and 12 are circuit schematics for a driving circuit for a unitcell in one embodiment of this invention. FIG. 12 expressly includestest circuitry, such as test transistors 320, 330, whereas FIG. 11 doesnot. The common aspects of the figures will be described together.

In one preferred embodiment of a unit cell 212, a symmetric arrangementis utilized. A first column select unit 260, preferably a transistor,and a first row select unit 270, also preferably a transistor, are inseries relation between a first source 284, e.g., voltage and/or currentsource, and a node 280, typically a current output node. In thepreferred embodiment, the column select transistor 300 may be preciselycontrolled under application of a gate voltage such as from the columnshift register memory (See FIG. 15). Preferably, the select units 260,260′ may differ from each other in their controllability, such as byvarying the channel length in the control transistor. Thus, byapplication of potentials from the row selector 220, 220′ and columnselector 230, 230′, application of potential to the control gates 302,312 results in output of current 282 at the unit cell.

The unit cell circuit 212 may further include a second column selectunit 270′, preferably a transistor 300′, and a second row select unit270′, also preferably a transistor 310′, used in series relation betweena second source 284′, e.g., voltage and/or current source, and a node,typically the previously referred to node 280, i.e., a current outputnode. In the preferred embodiment, the first source 284 is a supplypotential Vcc and the second source 284′ is a reference potential, suchas ground. Preferably the nodes are the same node 280, such that thereis a series connection between Vcc 284 and ground 284′ of the firstcolumn select unit 260, 260′ and first row select unit 270, the node280, and the second row select unit 270′ and the second column selectunit 260′.

In yet another form of operation of the circuit, or alternatively, adifferent mode of operation of the circuit shown in FIG. 11, the circuitmay be tested for continuity by simultaneously activating each of thefirst and second row and column select transistors 260, 270, 260′ and270. In this way the source 284 and sink 284′ are directly conductivelyconnected.

In yet another aspect of the preferred embodiment, test circuitry isincluded. FIG. 12 shows a schematic diagram of such a system. A firsttest transistor 320 spans the first column select transistor 260 andfirst row select 270 transistor. Likewise, a second test transistor 330spans the second column select transistor 260′ and second row selecttransistor 270′. Selective activation ensures continuity of the circuit.

While the circuitry described herein may be implemented in any knowntechnology consistent with the achievement of the desired functionalityof this system, one preferred mode of implementation is through CMOScircuitry. In one implementation of the circuits of FIGS. 11 and 12, thecolumn select devices 260, 260′ include a relatively long channellength. These relatively large field effect transistors serve to providemore accurate current control. By way of example, one implementation ofthis circuitry is in transistors having a 6 micron channel width. Theupper column select unit 260 (controlled by VI_P_CSEL) has a channellength of 80 microns, and the lower column select unit 260′ (controlledby signal VIN_N_CSEL) has a channel length of 126 microns. Thedifference in channel lengths reflects the difference in mobility ofelectrons and holes, and seeks to balance these two devices. By way ofcomparison, the remaining devices in FIGS. 11 and 12 have a 6 micronchannel width, and a 4 micron channel length. Alternativeimplementations of the unit cell 212 include series connection oftransistors, including dedicated series selection transistors for a rowselect and column select, plus an additional transistor for output level(current or voltage) select. More broadly, any circuit which receivescite selection information (e.g., row and column select) and valueand/or polarity information and causes the outputting of the desiredcurrent or potential may be utilized.

FIG. 13 shows a schematic view of a current control system useful in theinventions disclosed herein. To the extent possible, the numberingconvention is consistent with those of other drawings. The circuitserves to receive an input current 340 which is selectively controllableso as to generate a voltage at node 342 which is in turn coupled to aline 234 (shown to be the column line 234) which in turn is coupled tothe control element 266 of the variable current control element 260. Thevariable current control element 260, row select switch 270, firstsupply voltage 284 and output current from node 280 are as describedpreviously. Similarly, a current controlled circuit may be utilized tocontrol a symmetric circuit (e.g., elements 260′ and 270′ of FIG. 10).

The input currents 340 are provided to control elements 344. Eachcurrent of a given subscript is provided to a control element 344 oflike subscript. The control element 344 serves to selectively providecurrent at the output 346. As shown, the current outputs 346 are summedsuch that the current at node 348 may be varied based upon the states ofthe switches or control elements 344 a-d. A voltage divider arrangementis then provided wherein a potential 350 is provided to resistor 352which connects to node 342. By supplying the current at node 348 to node342 and then through resistor 352, a variable voltage is provided atnode 342. Optionally, the resistor 342 may be a device which, such as atransistor, which is conductive only in the event that current will besupplied to node 348. Thus, in the event that each switch 344 a-d is toremain off, such a circuit would also not include the resistor 352 inany state of conduction in that event. (See FIG. 15 for a detailedimplementation).

FIG. 14 shows a detailed circuit diagram for a portion of the currentmirror for use in the system. Four identical circuits are shown in FIG.14, and the description with respect to one circuit will apply to allcircuits equally. A current node 400 couples to the output of a firsttransistor 402 and second series connected transistor 404 which is inturn connected to a first potential 406 (Vdd). Optionally, thetransistors 402, 404 are biased to or connected to the supply voltage406. The control gate 408 for the second series connected transistor 404is connected to the current node 400. The current node 400 is alsoconnected to the output (source or drain) of first transistor 402. Thecontrol gate 410 of the first control transistor 402 is controlled by asignal 412. The signal 412 serves as a select signal for the currentmirror. The select signal 412 is supplied to the current mirrors tocause selective provision of the current from current nodes 400 to thecolumn select circuitry.

FIG. 15 is a detailed circuit diagram of a column select circuit (See,e.g., column selector 230 in FIG. 9). A shift register arrangement isprovided by a series of flip-flops 420, the first of which receives asan input the input information (Q(0)), optionally inverted by inverter422. As explained in connection with FIG. 9, an optional output 229 maybe provided from the selector, e.g., shift register 230. As shown, twostages, each comprising four bits, is shown for a shift register. Inimplementation, a 20×20 matrix or array of unit cells would require 80bits in the shift register 126 if 4 bits are assigned to each column.The outputs 430 are provided as control signals to current controlcircuitry 432. As shown, the current control circuitry consists of aparallel arrangement of a first transistor 434 and second transistor436, of opposite conductivity type, having their control gates coupledto the signal 430 as supplied directly to the first transistor 434 andthrough an inverter 438 to the second transistor 436. In operation, thecurrent supplied to node 440 is then selectively passed to output node442 under control of the signal 430. The current at the output node 442is summed with the output currents from the three other control circuits434 for the column position, the summing occurring by or before node496.

The summed current at node 496 is passed to node 492 which may serve asa voltage tap for column line 434. Logic 440, here shown to be a NANDgate, receives as inputs the outputs of the inverters 438. The outputsof the inverters 438 are provided to the NAND gate 440, which logicallyserves as an OR for the various inputs. Thus, the selection of any ofthe various current sources serves to activate the gated transistorcontrolled by the logic element 440.

The shift register 126 includes multiple series connected flip-flops420. The value signal is provided as input to the inverter 422 and thento the D input of the flip-flop 420. A clock signal (CM) and chip selectsignal (CS) are provided. The output of the last flip-flop 420 (rightmost in FIG. 15) is provided to an inverter which provides the outputbit at node 424.

FIG. 16 shows a component level schematic for a shift register 450.Flip-flops 452 receive an input 454 (Q(0)) which is passed to otherflip-flops 452 via the Q output of one flip-flop to the D input of thenext flip-flop 452. Optionally, an output 456 provides an indication ofthe most significant bit (or other indicator of loading) from the shiftregister. The enable signal 460 is provided as input to logic 462 (shownhere to be a NAND gate) which also receives as input the output (Q pin)of the associated flip-flop 452. The output of logic 462 controls passcircuitry 464 which serves to selectively pass the signal 466 which ifpassed through circuitry 464 constitutes the row select signal 468. Thecircuitry repeats for the number of stages in the shift register 450,and the description provided here applies to those stages.

FIG. 17 shows the layout for one implementation of a unit cell. Columnlines 234, 234′ are shown running vertically, which couple to the columnselectors (See FIG. 9). Row lines 224, 224′ are shown runninghorizontally. The supply voltage VDD 500 and the second voltage 202 (VSSe.g., ground) are disposed running generally parallel to the columnlines, 234, 234′. The optional test control lines 504, 504′ providecontrol signals for the n test and p test circuitry, respectively. Rowline 224 is connected by conductive member 506 to gate 508 whichoverlies the channel region underneath. Likewise, the row line 224′couples to gate 508′ which overlies the channel region for the selecttransistor. The column lines 234, 234′ are electrically coupled to gates510, 510′ which overlie the channel region which are then coupled to thefirst supply voltage VDD 500 and second supply voltage VSS 502,respectively. The channel length underlying the gates 510, 510′ differ,the difference in length being selected such that the operative deviceshave similar suitable properties. The output of the switchingtransistors controlled by gates 508, 508′ are provided throughconductive member 512 to electrode 514.

FIG. 18 shows a plan view of a portion of a 20×20 array of unit cells.FIG. 18 shows a portion of the overall chip, recognizing that thestructures such as the unit cells, shift registers, row and columndecoders and current mirrors are typically repeated identicallythroughout the chip. A plurality of unit cells (shown in detail in FIG.17) are included. Counter or return electrodes 520 are preferablydisposed at the periphery of the array of the unit cells. The electrodes520 preferably encompass or circumscribe the unit cell array.Optionally, multiple electrodes may be utilized to encompass the array.In the preferred embodiment, 4 L-shaped electrodes bracket the array (acorner of one being shown), each electrode bracketing substantially ¼ ofthe array. These electrodes may be utilized to move undesired materials,and to serve as a dump or disposition electrode. Row selectors 220″ (solabeled to correspond to the numbering in FIG. 9) are disposed to theright of the array and electrode 520 in FIG. 18). Column selectors 230″are disposed exterior to the array and the electrode 520. Current mirrorcircuitry 240″ is optionally disposed at the comers of the chip. Theselection and arrangement of components on chip is made to optimize thefunctionality of the device. Inclusion of components on chip, which istypically the disposable component, permits local control offinctionality, though with increased device costs. While variousarrangements are possible, the structure shown in FIG. 18 is thepreferred embodiment for the 20×20 chip.

FIG. 19 is a schematic diagram of the overall system. A control computer530 is coupled to a test board 532 and probe card 534 via buses 540.Optionally, a connector, such as an RS232 connector is utilized. Theprobe card 534 interfaces with the actual active electronic device. Theoutput from the device may be provided to receiving systems 542, whichmay include analog to digital converters for provision of digital datavia the bus 540 to the computer system 530.

FIG. 20 is a expanded block diagram of the test board and probe card ofFIG. 19. The serial port connection 550 couples to a universalasynchronous receiver transmitter (UART) 552 onto a controller 554. Businterconnection then couples to current sources 556 and current sinks558. Various digital to analog converters such as the dump DACs(digital-to-analog converters) 560 and bias DAC 562 are provided. Shiftregisters 264 coupled to the probe card 534. Analog to digitalconverters 556 may receive an output signal, such as from the probe card534. If the shift registers include an output (See output 229, 229′,239, 239′ and FIG. 9) a shift register loopback 568 may be provided.

FIG. 21 shows a graph of electronic hybridization utilizing the chip ofFIG. 2. The graph shows the fluorescent intensity, in MFI/s as afunction of column number. The three bar graphs labeled column 1, column2 and column 3 utilize field shaping, and show specific hybridization onthe left bar graph in comparison to non-specific hybridization on theadjacent right hand column. The three couplets of bar graphs labeledcolumn 1, column 2 and column 3 above the designator “standard” show thesame system but without field shaping. The discrimination betweenspecific versus non-specific binding is significantly less than in thecase where field shaping is utilized. The sequences wereATA5/ATA7/biotin, and 10 pM RCA5/BTR.

FIG. 22 shows a graph of experiments performed with the system as shownin FIG. 2. The y-axis shows the average MFI/second, and the x-axis showsvarious rows of various concentrations. The first couplet of paragraphsshows a 50 nM concentration of RCA5 BTR reporter in 50 mM histidine, anddepicting the specific/non-specific binding after washing. The firstcouplet shows rows 1 and 2 comparing the specific binding (ATA5/RCA5) tothe non-specific binding (ATA7/RCA5), showing a 12:1 and 50:1improvement. The middle couplets of bar graphs show a 50 pMconcentration of RCA5 BTR reporter and shows a 3.9:1 and 4.9:1 ratio ofspecific binding to non-specific binding signal intensity. The last setof couplet bar graphs shows a 1 pM concentration of RCA5 BTR reporterand shows a 4.4:1 and 4.0:1 ratio of specific binding to non-specificbinding.

FIG. 23 is a graph of current linearity showing the electrode currentoutput in nanoamps as a function of current input in microamps. A legendis provided to indicate the various lines on the graph.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spirtor scope of the appended claims.

We claim:
 1. A method for manufacturing a flip-chip device forperforming active biological operations, the device including asubstrate having first and second surfaces, the substrate including avia between the first and second surfaces, the substrate furtherincluding electrically conductive traces on at least one of the firstand second surfaces, the device further including a chip having firstand second surfaces, the chip including electrically conductive traceson the first surface connecting to an array of microlocations, the arrayof microlocations adapted to receive a fluid placed upon the substrateand through the via, the array of microelectrodes being disposed withina sealant free reaion on the first surface of the chip, the methodcomprising the steps of: placing the chip adjacent to the substrate;forming electrically conductive interconnects, interconnecting theelectrically conductive traces on the first surface of the chip with theelectrically conductive traces on either the first or second surfaces ofthe substrate; exposing light onto the substrate and through the via,down to the first surface of the chip; and applying a light curable,wickable sealant to the interface between the substrate and the chip,said interface between the substrate and the chip lying outside thesealant free region, wherein the light at least partially cures thesealant, whereby the sealant is precluded from flowing to the sealantfree region so as to leave the array of microlocations free of sealant.2. A method according to claim 1, wherein the electrically conductiveinterconnects are bumps.
 3. A method according to claim 1 furtherincluding the step of completing the cure of the sealant.
 4. A methodaccording to claim 1, wherein the light exposed to the substrate is UVlight.
 5. A method according to claim 3, wherein the step of completingthe cure of the sealant is done by heat treatment.
 6. A method accordingto claim 1, wherein the sealant is applied to the interface between thechip and substrate by wicking.
 7. A method according to claim 1 furtherincluding the step of placing a flow cell on either the first or secondsurfaces of the substrate, the flow cell residing on a surface of thesubstrate opposite to the surface of the substrate adjacent to the chip.8. A method for manufacturing a flip-chip device for performing activebiological operations, the device including a substrate having first andsecond surfaces, the substrate including a via between the first andsecond surfaces, the substrate further including electrically conductivetraces on at least one of the first and second surfaces, the devicefurther including a chip having first and second surfaces, the chipincluding electrically conductive traces on the first surface connectingto an array of microlocations, the array of microlocations adapted toreceive a fluid placed upon the substrate and through the via, the arrayof microelectrodes being disposed within a sealant free region on thefirst surface of the chip, the method comprising the steps of: placingthe chip adjacent to the substrate; forming electrically conductiveinterconnects, interconnecting the electrically conductive traces on thefirst surface of the chip with the electrically conductive traces oneither the first or second surfaces of the substrate; applying acurable, wickable sealant to the interface between the substrate and thechip, said interface between the substrate and the chip lying outsidethe sealant free region, said applied sealant being of a sufficientviscosity and amount such that said sealant is precluded from flowing tothe sealant free region so as to leave the array of microlocations freeof sealant; and completing the cure of said sealant.
 9. A methodaccording to claim 8, wherein the electrically conductive interconnectsare bumps.
 10. A method according to claim 8, wherein the step ofcompleting the cure of the sealant is done by heat treatment.
 11. Amethod according to claim 8, wherein the sealant is applied to theinterface between the chip and substrate by wicking.
 12. A methodaccording to claim 8 further including the step of placing a flow cellon either the first or second surfaces of the substrate, the flow cellresiding on a surface of the substrate opposite to the surface of thesubstrate adjacent to the chip.
 13. A method for manufacturing aflip-chip device for performing active biological operations, the deviceincluding a substrate having first and second surfaces, the substrateincluding a via between the first and second surfaces, the substratefurther including electrically conductive traces on at least one of thefirst and second surfaces, the device further including a chip havingfirst and second surfaces, the chip including electrically conductivetraces on the first surface connecting to an array of microlocations,the array of microlocations adapted to receive a fluid placed upon thesubstrate and through the via, the array of microelectrodes beingdisposed within a sealant free region on the first surface of the chip,the method comprising the steps of: placing the chip adjacent to thesubstrate; forming electrically conductive interconnects,interconnecting the electrically conductive traces on the first surfaceof the chip with the electrically conductive traces on either the firstor second surfaces of the substrate; affixing an illumination member tothe substrate such that the illumination member is disposed adjacent toand substantially coplanar with the first surface of the chip; exposinglight onto the substrate and through the via, down to the first surfaceof the chip; and applying a light curable, wickable sealant to theinterface between the substrate and the chip, said interface between thesubstrate and the chip lying outside the sealant free region, whereinthe light at least partially cures the sealant, whereby the sealant isprecluded from flowing to the sealant free region so as to leave thearray of microlocations free of sealant.
 14. A method according to claim13, wherein the electrically conductive interconnects are bumps.
 15. Amethod according to claim 13 further including the step of completingthe cure of the sealant.
 16. A method according to claim 13, wherein thelight exposed to the substrate is UV light.
 17. A method according toclaim 15, wherein the step of completing the cure of the sealant is doneby heat treatment.
 18. A method according to claim 13, wherein thesealant is applied to the interface between the chip and substrate bywicking.
 19. A method for manufacturing a flip-chip device forperforming active biological operations, the device including asubstrate having first and second surfaces, the substrate including avia between the first and second surfaces, the substrate furtherincluding electrically conductive traces on at least one of the firstand second surfaces, the device further including a chip having firstand second surfaces, the chip including electrically conductive traceson the first surface connecting to an array of microlocations, the arrayof microlocations adapted to receive a fluid placed upon the substrateand through the via, the array of microlocations being disposed within asealant free region on the first surface of the chip, the methodcomprising the steps of: placing the chip adjacent to the substrate;forming electrically conductive interconnects, interconnecting theelectrically conductive traces on the first surface of the chip with theelectrically conductive traces on either the first or second surfaces ofthe substrate; affixing an illumination member to the substrate suchthat the illumination member is disposed adjacent to and substantiallycoplanar with the first surface of the chip; applying a curable,wickable sealant to the interface between the substrate and the chip,the interface between the substrate and the chip lying outside thesealant free region, said applied sealant being of a sufficientviscosity and amount such that said sealant is precluded from flowing tothe sealant free region so as to leave the array of microlocations freeof sealant; and completing the cure of said sealant.
 20. A methodaccording to claim 19, wherein the electrically conductive interconnectsare bumps.
 21. A method according to claim 19, wherein the step ofcompleting the cure of the sealant is done by heat treatment.
 22. Amethod according to claim 19, wherein the sealant is applied to theinterface between the chip and substrate by wicking.
 23. A method formanufacturing a multicomponent flip-chip device for performing activebiological operations, the device including a chip disposed adjacent toa substrate, the substrate including a via therethrough, wherein thedevice is adapted to receive a fluid to be placed on the substrate, andto flow through the via down to the chip, the method comprising theconsecutively performed steps of: placing a chip adjacent to asubstrate; applying a curable, wickable sealant to the interface betweenthe substrate and the chip, said applied sealant being of a sufficientviscosity and amount such that said sealant is precluded from flowing toat least a portion of the chip bounded by the via; and completing thecure of said sealant by heat treatment.
 24. A method for manufacturing amulticomponent flip-chip device for performing active biologicaloperations, the device including a chip disposed adjacent to asubstrate, the substrate including a via therethrough, wherein thedevice is adapted to receive a fluid to be placed on the substrate, andto flow through the via down to the chip, the method comprising theconsecutively performed steps of: placing a chip adjacent to asubstrate; wicking a curable, wickable sealant to the interface betweenthe substrate and the chip, said sealant being of a sufficient viscosityand amount such that said sealant is precluded from flowing to at leasta portion of the chip bounded by the via; and completing the cure ofsaid sealant.